Muzolimine: renal site of action and interaction with probenecid in humans.

Muzolimine (60 mg, administered orally) was administered to eight healthy volunteers, under conditions of altered fluid load, to elucidate its renal site of action. The duration of action and the effect of probenecid pretreatment on muzolimine response was also investigated. Muzolimine had a rapid onset of action, with the diuresis complete within 4 hours after dosing. At peak natriuresis, under hydrated conditions, fractional excretion of free water remained unaltered (9.72% +/- 0.59% versus 9.07% +/- 0.44%; difference not significant) but was accompanied by a significant increase in the delivery of sodium out of the proximal tubule, as measured by fractional excretion of lithium (22% +/- 2% to 31% +/- 1%; p less than 0.01). The fraction of sodium reabsorbed in the distal tubule also decreased from 94% +/- 1% to 67% +/- 1% (p less than 0.001) of the delivered load. The fractional reabsorption of free water during hydropenia decreased after muzolimine (5.63% +/- 0.26% to 2.00% +/- 0.81%; p less than 0.05). Pretreatment with probenecid resulted in a prominent decrease in urinary sodium excretion (246 +/- 25 mmol/24 hr for muzolimine alone 161 +/- 24 mmol/24 hr for muzolimine and probenecid; p less than 0.01). These findings suggest that muzolimine has a major site of action in the medullary portion of the thick ascending limb of Henle with additional inhibitory activity on the proximal tubule. It is likely that the active secretion of one or more of the acidic metabolites of muzolimine, by way of the probenecid sensitive organic acid pathway, is responsible for mediating the renal actions this basic drug.

Mechanisms of the inhibition of human erythrocyte pyridoxal kinase by drugs.

The aim of this study was to investigate the interaction between drugs chosen for their clinical neurotoxicity or chemical structure and vitamin B6 metabolism. After a preliminary screening of drugs to determine their potential inhibitory effect on erythrocyte nonpurified pyridoxal kinase (PLK) (EC 2.7.1.35), additional investigations, including kinetic studies and detection of chemical reactivity between the inhibiting drugs and pyridoxal (PL) or pyridoxal-5'-phosphate (PLP), using UV-visible spectrophotometry and mass analysis, were carried out to specify the mechanism of PLK inhibition. Depending on the results, the inhibiting drugs were divided into three groups. The first group included theophylline and progabide and inhibited PLK using either PL or pyridoxamine (PM) as substrate and thereby were true inhibitors. Moreover, they did not form covalent complexes with PL or PLP. The second group, which included cycloserine, dopamine, isoniazid, and thiamphenicol glycinate, inhibited PLK using PL, but not PM, as substrate. They were able to react with PL or PLP to form covalent complexes, and kinetic studies suggested that the observed PLK inhibition was due to these formed complexes. A third group, which consisted of levodopa, D-penicillamine, and muzolimine, inhibited PLK using PL, but not PM, as substrate. They formed, with PL or PLP, chemical derivatives that probably had no inhibitory effect on PLK. These results and the clinical consequences of such interactions are discussed and compared with results of previous studies.

Influence of muzolimine on arterial wall elastin.

Muzolimine, 3-amino-1-(3,4-dichloro-alpha-methylbenzyl)-2- pyrazolin -5-one, an antihypertensive and diuretic drug, accumulates in the arterial tissue of rats and dogs after oral administration. Two weeks after the administration of 3 mg [14C]muzolimine, the aorta of rats contained 60-300 times more 14C-radioactivity/weight unit than the skin or tail tendon. The 14C-radioactivity was exclusively bound to the isolated aortic elastin and corresponded to 0.04% of the applied muzolimine dose. Up to ca 250 ng bound muzolimine/mg elastin was found in the aorta of dogs treated with non-labelled muzolimine for 52 weeks. The elastin-bound [14C]muzolimine was not extractable by organic solvents or by weak acids or bases but was released in a soluble form by pancreatic elastase and extracted from the elastase digest by dichloromethane. In the dichloromethane extract muzolimine was detected by HPLC and HPTLC, and was identified by mass spectrometry. Muzolimine pretreatment of rats for 2 months did not influence the elastin content of arterial tissue or [3H]glycine incorporation into aortic elastin under organ culture conditions, but after labelling the elastin with [4,5-3H]lysine, the [3H]desmosine and [3H]-isodesmosine isolated from the elastin of muzolimine-pretreated rats and incorporated under organ culture conditions was lower than that of control animals. In addition, aortic elastin of rats pretreated for 2 months with 800 ppm muzolimine in the diet was more resistant to elastase degradation. This effect might give some implications for muzolimine in the therapy of cardiovascular disorders with impaired arterial elastin metabolism.

The effects of muzolimine and urine from muzolimine-treated rats on Na+K+Cl- cotransport in Madin-Darby canine kidney cells.

Muzolimine is a loop diuretic with an original chemical structure devoid of the acidic or sulfonamide group known to be necessary for an interaction with Na+K+Cl- cotransport. We studied the effects of urine from muzolimine-treated rats on the Na+K+Cl- cotransport-dependent 86Rb influx in MDCK cells. Na+K+Cl- cotransport was inhibited by urine obtained 15 min (42% inhibition) and 60 min (49% inhibition) after muzolimine injection (50 mumol/kg i.v.). Muzolimine itself was not detectable in the urine. Probenecid (100 mumol/kg i.v.) suppressed both the diuretic effect of muzolimine and the inhibition of Na+K+Cl- cotransport by urine from muzolimine-treated rats. These results suggest that the diuretic effect of muzolimine is due to the metabolism of muzolimine into an active compound which inhibits Na+K+Cl- cotransport after its secretion into the tubular lumen via a proximal pathway. The direct effect of muzolimine on Na+K+Cl- cotransport in MDCK cells was also tested: surprisingly, the inhibition of 86Rb influx was significant in the presence of muzolimine (IC50 = 1.44 microM). We show that this effect was due to the metabolism of muzolimine by these cells into an active compound.

Inhibition of the Cl-/NaCO3- anion exchanger by xipamide in human red blood cells.

The mechanism of action of classical loop diuretics of the 2- or 3-amino-5-sulfamoylbenzoic acid and (aryloxy)acetic acid families involves competition with chloride for a common site on the (Na+, K+, 2Cl-) co-transport system. However this is not the mechanism of action of some high-ceiling diuretics like muzolimine, MK 473, xipamide, indapamide and clopamide, which are not carboxylic acids. We evaluated three of these latter diuretics (xipamide, muzolimine and clopamide) for their inhibitory effects on five ion transport systems in human red blood cells: (i) Cl(-)-dependent (Na+, K+) co-transport, (ii) (NaCO3-/Cl-) anion exchanger, (iii) (Cl-, K+) co-transport, (iv) Na+, K+ pump and (v) Na+: Li+ counter-transport; and on one ion channel the Ca2+-dependent, K+ channel. All erythrocyte transport pathways were resistant to the three diuretics studied (IC50 of 10(-3) M or higher) with one remarkable exception, the (NaCO3-/Cl-) anion exchanger. This transport system was inhibited by xipamide (IC50 of 2.5 +/- 0.4 X 10(-5) M, mean +/- S.D. of five experiments) and less potently by muzolimine (IC50 of 1.1 +/- 0.3 X 10(-4) M, mean +/- S.D. of three experiments). Clopamide only inhibited the anion exchanger at high concentrations (IC50 of about 10(-3) M). Xipamide, the most potent diuretic in this test, was at least one order of magnitude more active than furosemide, ethacrynic acid, hydrochlorothiazide and amiloride. Inhibition of the anion carrier could be involved in the diuretic action (inhibition of CO2-stimulated NaCl absorption in the TAL) and/or in the antihypertensive action (inhibition of net NaCO3- influx and secondarily of Ca2+ influx through Na+: Ca2+ exchange in vascular smooth muscle cells of xipamide).

Inhibition of ion transport in Ehrlich cells by muzolimine.

The influence of muzolimine on the transport of Na+, K+ and Cl- was studied in Ehrlich cells to test whether the diuretic inhibits the furosemide-sensitive Na+-K+-2 Cl-(-)cotransport or transport via the ouabain-sensitive Na+/K+-pump. It was shown that between 10(-5) M and 10(-3) M muzolimine pump-flux decreases with increasing drug concentration (IC50 about 0.5 mM), in contrast to the unaffected cotransport. This reduction in pump rate is only seen with respiring cells, but not during glycolytic ATP-production. Therefore, muzolimine seems to inhibit the Na+/K+-pump not directly but indirectly by interference with energy metabolism resulting in decreased ATP concentration. This reduction in ATP-level is at least partially due to activation of an ATP-consuming process of unknown nature. Whether muzolimine also inhibits respiration was not tested.

Clinical trial with muzolimine in healthy and hypertensive subjects: new vistas on the drug action.

The mechanism of muzolimine (3-amino-1-[3,4-dichloro-alpha-methyl-benzyl]-2 pyrazolin-5-one) action is still not completely defined. The identified site of action is the Henle loop, similarly to furosemide which acts also by mediating renal prostaglandin synthesis. The aim of the present study was to evaluate the early effects of muzolimine (30 mg per os) on renal function and prostaglandin urinary excretion in healthy controls and hypertensive subjects. Urinary flow reached the peak values by the third hour after the drug and a diuretic effect not directly dependent on glomerular filtration was observed, especially in hypertensive patients. In these cases the diuresis increased also due to a low glomerular filtration rate and tubular phenomena were more evident than in controls: an increasing Na+ tubular excretion and a parallel decreasing % Na+ reabsorption. Blood pressure was not significantly influenced by muzolimine in healthy subjects, while it returned to normal values in the hypertensive group. A cyclooxigenase inhibitor, lysine acetylsalicylate (1 g i.m.) administered 10 minutes after muzolimine, was not able to modify the parameters under consideration. Therefore a mediation by prostaglandins on the diuretic and antihypertensive effects of the drug under study may probably be excluded.

[Mechanism of action of the diuretic effect of muzolimine]. / Mécanisme d'action de l'effet diurétique de la muzolimine.

Muzolimine is a diuretic which has been proposed in the treatment of hypertension. Muzolimine shared both the high ceiling effect of loop diuretics and the long duration of action of thiazides but has a chemical structure different from those of other loop diuretics. It may act as a prodrug and an active metabolite present in the urine may inhibit NaCl reabsorption in the Henle's loop. We studied the effect of urines of piretanide and muzolimine treated rats on Na+K+Cl- cotransport in renal cells in culture (MDCK). In the presence of ouabain (0.5 mM), the Na+K+Cl- cotransport measured by 86Rb influx, represented 92 p.100 of the total 86Rb influx (6.16 +/- 1.12 nmol 86Rb/min/mg prot, n = 10). Both diuretics were administered i.v. to rats where they induced marked diuresis. Excreted urine (dilution to 1/100) was tested for cotransport inhibition. After piretanide (27 mumol/kg) the urine inhibited the Na+K+Cl- cotransport in MDCK cells (72% and 41% inhibition at the 15th and 45th minutes after diuretic injection). After muzolimine (50 mumol/kg), urines also inhibited Na+K+Cl- cotransport but the effect was slower in onset and more prolonged (42% and 49% inhibition at the 15th and 60th min). Diuretic effects in vivo and Na+K+Cl- cotransport inhibition in vitro by the urine developed parallel for both diuretics. Probenecid (100 mumol/kg) suppressed simultaneously the diuretic effect of muzolimine and the Na+K+Cl- cotransport inhibition by the urine of muzolimine treated rats. Our results suggest that muzolimine acts as a prodrug. Its active metabolite is secreted into the tubular lumen through a probenecid sensitive pathway and inhibits, like other loop diuretics, Na+K+Cl- cotransport.

Suppression of renin-angiotensin system in the heart of spontaneously hypertensive rats.

Renin-like enzyme and angiotensin converting enzyme (ACE) were identified and their specific activities measured in cardiac tissues of spontaneously hypertensive rats (SHR) and their Wistar-Kyoto (WKY) normotensive controls. In addition, the enzyme activities were determined following administration of hypotensive drugs. The pH optima of cardiac renin-like enzymes were identical with those in vascular walls, the specific activity being higher in the heart. Cardiac ACE revealed similarities with the venous wall enzyme. The highest specific cardiac renin-like activity was found in the septum and that of ACE in atria/auricles. Both enzyme values were lower in the hearts of SHR than in those of normotensive controls. Following nifedipine treatment, specific renin-like activities increased in all cardiac structures studied (P less than 0.01); with nitrendipine and muzolimine less pronounced elevations were obtained. Administration of these three hypotensive drugs resulted in a stimulation of ACE in all the cardiac structures except in atria/auricles, where their activities were lowered.

Changes of electrolyte excretion, renin activity, angiotensin-II and aldosterone concentrations during administration of 3-amino-1-(3,4-dichloro-a-methyl-benzyl)-2-pyrazolin-5-one (Bay g 2821).

After the administration of 40 mg Bay g 2821, a new potent diuretic substance, a significant increase of sodium and water diuresis occurred. There was only a short rise in potassium excretion. In contrast to the sodium and water diuresis no significant increase of renin-activity, angiotensin-II or aldosterone concentration was seen. In spite of the insignificant stimulation of the renin-angiotensin-aldosterone system, a significant increase of correlation occurred between the three components of the renin-angiotensin-aldosterone system.

The effects of single doses of muzolimine upon urinary solute and fluid excretion.

The natriuretic effect of many loop diuretics is followed by an important decrease - rebound undershoot - in renal Na+ excretion, which is accompanied by thirst and fluid reposition. Also most loop diuretics increase K+ and Mg2+ urinary outputs, thus leading to somatic depletion of these cations and subsequent cardiac arrhythmias. The objectives of the present study were to describe the urinary outputs and flows of several solutes after various doses of muzolimine. Experiments were carried out in ten healthy adult volunteers given monodoses of placebo and of muzolimine 20, 30 and 40 mg on separate days and in random order. Urine collected at 3, 6, 9, 12 and 24 h after dosing was analysed for solutes. Muzolimine 20 mg did not increase mean 24 h urinary Na+ output significantly with respect to placebo. Muzolimine 30 mg exerted maximal diuretic, chloriuretic and natriuretic effects, while not affecting the mean 24 h urinary outputs of K+ and Mg2+ significantly. Muzolimine 30 mg caused a small undershoot in urinary Na+ flow after completion of its natriuretic effect. Muzolimine 30 mg should be considered a first choice loop diuretic since it only causes a mild post-natriuretic undershoot and does not increase urinary Mg2+ output significantly.

Clinicopharmacological reappraisal of the potency of diuretics.

From a clinicopharmacological standpoint, the urinary excretory potency of diuretics should be assessed comparatively on the basis of the changes in 24-hour natriuresis, with respect to 24-hour natriuresis after placebo, caused by single oral doses administered to healthy adult subjects who are in habitual and steady-state external sodium balance. The potency of various formulations of loop (e.g., furosemide), of early distal tubular (e.g., the thiazides), and of potassium-retaining diuretics, as well as of several combinations of diuretics, has been evaluated in a series of studies. Two formulations of loop diuretics (muzolimine 20 mg and torasemide 2.5 mg) are definitely nondiuretic. The majority of the other formulations of loop diuretics studied are, in general, comparatively less potent than most of the common formulations of early distal tubular diuretics studied. As a general rule, most common formulations of early distal tubular diuretics are at least not less potent than the majority of common formulations of loop diuretics. Hydrochlorothiazide 25 mg and furosemide 80 mg have similar potencies. Loop diuretics increase mean renal sodium output strikingly within the first few (0-6) hours after dosing, but this forced excretion is followed by a rebound with respect to postplacebo mean urinary sodium flow; the rebound usually takes place between 6 and 24 hours after dosing. However, no rebound in mean urinary sodium flow occurs during the 24 hours following a single dose of a distal tubular diuretic; these substances increase urinary sodium excretion with lower maximal intensity but more protractedly than loop diuretics.(ABSTRACT TRUNCATED AT 250 WORDS)

[Can gentamicin nephrotoxicity in rats be modified by chronic administration of muzolimine?]. / La néphrotoxicité de la gentamicine peut-elle être modifiée chez le rat par l'administration chronique de muzolimine?

Some loop diuretics seem to increase gentamicin nephrotoxicity. We investigated this property for a new diuretic, muzolimine, in female Wistar rats. Each rat was given gentamicin intraperitoneally in a dosage that induces morphological and functional modifications in the kidneys (20 mg/kg/day for 7 days). Muzolimine was given in a daily dosage of 15 mg/kg starting 15 days before the first gentamicin injection and continuing throughout the seven-day gentamicin course. As compared to controls given gentamicin alone, modifications induced by the muzolimine-gentamicin combination showed no significant differences for the following criteria: renal accumulation of gentamicin, membrane lysosomal latency, renal oxidative mitochondrial metabolism and cortical activity of renal cathepsin B, N-acetyl-beta-D-glucosaminidase (NAG) and alanine aminopeptidase. However, slightly lower sphingomyelinase activities (p less than 0.05) were found for muzolimine + gentamicin as compared to gentamicin alone. Urinary NAG excretion increase more with muzolimine + gentamicin than with gentamicin alone, perhaps as a result of the very significant increase in urinary output observed with the diuretic. Our results show that, in contrast to findings with another loop diuretic, furosemide, the renal toxicity of gentamicin is not noticeably modified by concomitant administration of muzolimine.

Demonstration of action of muzolimine on the serosal side of Henle's loop cells.

Muzolimine, a new diuretic, is known to act by inhibiting Na transport along the ascending limb of Henle's loop. Because of its ineffectiveness when perfused into the lumen, and as a result of experiments on frog skin, it is believed that muzolimine acts from the serosal cell side, unlike all other diuretics. We decided to assess the site of action of muzolimine (mucosa versus serosa) by experiments on rats. The animals were anesthetized, both ureters cannulated, and a hydronephrosis established in the left kidney by applying a counterpressure to the ureter, slowly and progressively increased up to 60 cm H2O in 3 minutes and kept stable for 20 minutes. Then either muzolimine 1.2 mg, or furosemide 4 mg, was injected i.v. together with 131I-Hypaque as a glomerular marker. Three minutes later the hydronephrosis was released in the left kidney by cutting the catheter, and urine collections were started simultaneously in both kidneys into 25 microliter glass capillaries, that were filled in continuous sequence and numbered progressively. In each urine sample from each glass capillary Na and 131I-Hypaque were measured. In 5 animals receiving furosemide, UNa rose in the left post-obstructed kidney and reached a plateau level by the 3rd to 5th sample. The rise in Na concentration, indicating delivery of urine whose Na reabsorption had been inhibited along the loop of Henle paralleled that in 131I-Hypaque, indicating that the diuretic and the glomerular marker had reached Henle's loop simultaneously, following filtration after the hydronephrosis had been discontinued.(ABSTRACT TRUNCATED AT 250 WORDS)

Acute and chronic diuretic-induced alterations in urinary sodium and calcium excretion in the conscious rat.

The acute and chronic (5-day) effects of three low-ceiling diuretics (hydrochlorothiazide, indapamide or xipamide) and two high-ceiling diuretics (furosemide or muzolimine) on urinary volume and electrolyte excretion were examined in the saline-loaded conscious rat. The three low-ceiling agents produced qualitatively similar alterations in both acute and chronic volume and electrolyte excretion patterns. Each of these agents produced an acute disassociation between urinary sodium and calcium excretion and hypocalciuria after chronic administration. It is suggested that indapamide and xipamide, like hydrochlorothiazide, inhibit sodium but not calcium reabsorption in the distal nephron. Acute as well as chronic treatment with either furosemide or muzolimine produced dose-dependent increases in urinary volume and electrolyte excretion. A generally parallel response was observed on sodium and calcium excretion with these agents. It is suggested that muzolimine, like furosemide, inhibits both sodium and calcium reabsorption in the ascending limb of Henle's loop.

Influence of muzolimine, a new diuretic, on experimental gentamicin nephrotoxicity.

The administration of diuretic drugs increases the nephrotoxicity of aminoglycosides. Muzolimine is a new diuretic which acts on the ascending limb of the loop of Henle and on the distal tubule. It differs from frusemide in its strong lipid binding and in its action at the antiluminal cellular pole. We attempted to determine whether muzolimine increases the nephrotoxicity of gentamicin. Four groups of Wistar rats were studied: one control group, one group treated with muzolimine (15 mg/kg), one group treated with gentamicin (20 mg/kg), and one group treated with gentamicin and muzolimine. Muzolimine induced a moderate but significant decrease in creatinine clearance, increased urinary excretion of n-acetyl-beta-D-glucosaminidase, and a slight decrease in lysosomal latency. The action of muzolimine did not reduce enzyme activities of the renal cortex (AAP, NAG, sphingomyelinase). Gentamicin induced functional renal modifications known to characterise nephrotoxicity of aminoglycosides. The association of muzolimine and gentamicin did not provoke a greater decrease of creatinine clearance or a greater decrease in enzyme activities of the renal cortex. In conclusion, muzolimine does not seem to potentiate the nephrotoxic action of gentamicin. However, we note that the association of muzolimine and gentamicin provoked a greater decrease in mitochondrial oxygen consumption than that provoked by gentamicin alone. It also induced more marked reduction of the activity of renal sphingomyelinase. This point is important when it is considered that the appearance of myelin bodies in the renal cortex is secondary to the decrease of this enzyme.

The diuretic effect of muzolimine.

Muzolimine produces a diuresis in the loop of Henle. Unlike other diuretics of this kind, the effect of muzolimine is slow and its action is long lasting. The present study was designed to examine the mechanism of action of muzolimine in isolated in vitro perfused rabbit cortical thick ascending limb segments (cTAL). In a first series it was confirmed that muzolimine itself in a concentration of up to 10(-4) mol/l had no effect on the equivalent short circuit current (Isc), corresponding to the rate of active NaCl reabsorption, in cTAL segments neither from the lumen nor from the bath side. In a second series of experiments, muzolimine was administered intravenously (70 mumol/kg) to male Wistar rats, and the clearances of inulin, Na+ and K+ were calculated. Muzolimine, after a lag phase of 5 to 20 min caused a marked diuresis and natriuresis, but had only a minor effect on glomerular filtration rate and on K+ excretion. The urines of these animals were diluted with isotonic saline, and were examined in rabbit cTAL segments for their ability to block Isc. It was found that the urines of the antidiuretic period were devoid of effects, whereas the diuretic urines inhibited Isc strongly down to a dilution of 300 times when they were perfused in the lumen. The same diluted urines had no effect from the bath side. In a third series, probenecid (100 mumol/kg) was administered i.v. to rats undergoing a muzolimine induced saluresis. It was found that probenecid inhibited the diuresis and saluresis.(ABSTRACT TRUNCATED AT 250 WORDS)

Antagonistic effects of guanabenz, carteolol, and muzolimine on hypertensive responses in the anesthetized dog.

In dogs, treatment with guanabenz, carteolol, and muzolimine for 7 days, reduced the blood pressure responses to bilateral occlusion of the carotid arteries, electric stimulation of central vagus nerve, acetylcholine after atropinization, nicotine, l-noradrenaline, angiotensin II, l-adrenaline, KC1 and asphyxia.